Method of treating obesity

ABSTRACT

The inhibition of fatty acid synthesis is obtained in biological systems by utilizing a specific stereoisomer of hydroxycitric acid and derivatives thereof such as esters or lactones and the non-toxic salts of these compounds. It is believed that the present method involves the inhibition of citrate cleavage enzyme. Inhibition of fatty acid synthesis by the present method is useful in the treatment of obesity.

U 4 Unite States Patent [1 1 [111 3,764,692 Lowenstein Oct. 9, 1973METHOD OF TREATING OBESITY Chemical Abstracts 65: 9373 a [75] Inventor:John M. Lowenstein, Wellesley Chemical Abstracts 67: 69394 G Hills,Mass. Chemical Abstracts 70: 105772 b [73] Assignee: Hoffmann-La RocheInc., Nutley, Merck Manual 11th Edmon 1966 307-311 NJ. PrimaryExaminer-Albert T. Meyers Filed? p 1970 Assistant Examiner-Norman A.Drezin App]. No.: 77,042

Related (1.8. Application Data Continuation-impart of Ser. No. 872413,Oct. 29, 1969, abandoned.

US. Cl .i 424/279, 424/313, 424/317 Int. Cl A61k 27/00 Field of Search424/317, 279, 313

References Cited OTHER PUBLICATIONS Chemical Abstracts 60: 13800 bChemical Abstracts 63: 16775 g Att0rneySamuel L. Welt, Jon S. Saxe,Bernard S. Leon, William H. Epstein and George M. Gould [57] ABSTRACT 12Claims, N0 Drawings METHOD OF TREATING OBESITY RELATED APPLICATIONS Thisapplication is a continuation-in-part of applicant's copending US. Pat.application Ser. No. 872,4- 13 filed Oct. 29, 1969, now abandoned.

BRIEF DESCRIPTION OF THE INVENTION This invention relates to a method ofinhibiting fatty acid synthesis in biological systems by introducinginto such systems a specific stereoisomer of hydroxycitric acid orderivatives thereof. The biological systems in which the method of thepresent invention may be practiced include cell free enzyme preparationscontaining citrate cleavage enzyme (also identified as ATPzcitrateoxaloacetate lyase), citrate, coenzyme A, ATP (or systems generatingATP), TPNI-I (or systems generating TPNH), and tissue homogenates,tissue slices, perfused organs, and intact mammals, particularlynon-ruminating mammals.

The stereoisomers of hydroxycitric acid and its derivatives are relatedstructurally to citric acid wherein a hydroxy group is substituted forone of the four methylene hydrogens of citric acid. Thus, there are fourpossible stereoisomers of hydroxycitric acid. Of these fourstereoisomers one has been found to inhibit substantially fatty acidsynthesis in biological systems. This particular isomer is(-)hydroxycitric acid hereinafter called garcinia acid. It is obtainableby isolation from the fruit of Garcinia cambogia using known procedures.For example, this isolation may be accomplished following the proceduredescribed by Lewis in Methods in Enzymology (J. M. Lowenstein, ed.),Vol. 13, page 613 (Academic Press, New York, 1969).

Garcinia acid is usually isolated in the form of its lactone. The freeacid may be conveniently obtained from the lactone by base hydrolysis,e.g., sodium hydroxide or potassium hydroxide preferably with heatingfollowed by acidification in a manner known per se.

The term derivatives" as used herein in conjunction with garcinia acidis meant to include garcinia acid lactone, derivatives of one or morecarboxyl groups of garcinia acid, e.g., mono, di or tri esters ofgarcinia acid or mono or di esters of its lactone and non-toxicpharmaceutically acceptable basic salts of garcinia acid or the lactoneor esters thereof.

Ester derivatives of garcinia acid which are useful in the practice ofthe present invention include the lower alkyl, aryl and aryl-lower alkylesters. Included within the lower alkyl esters of the present inventionare branched or straight chain lower alkyl radicals having from one toseven carbon atoms. Preferred lower alkyl esters of garcinia acidinclude the methyl, ethyl, isopropyl and butyl esters. Examples of arylesters include the phenyl and substituted phenyl esters, e.g., phenylsubstituted with halogen, lower alkyl, lower alkoxy or nitro. Benzylrepresents a preferred aryl alkyl ester. The aforesaid esters may beprepared by esterification of garcinia acid with a desired alcohol inthe presence of excess mineral acid such as sulfuric acid, hydrobromicacid, or the like. Suitable alcohols include lower alkanols, phenol andbenzyl alcohol, for example. Conventional esterification conditions maybe employed. Additionally, alkyl or aralkyl esters may be prepared byreaction with diazoalkylenes or diazoarylalkylenes, e.g., diazomethane,diazoethane or phenyldiazomethane in a manner known per se.

The garcinia acid may also be utilized in the form of itspharmaceutically acceptable non-toxic basic salt. Preferred salts forthis purpose include the alkali metals, e.g., sodium or potassium; thealkaline earth metals, e.g., calcium; or complex salts such as ammoniumor substituted ammonium salts such as a mono-, dior tri-alkylammoniumsalt or a mono-, dior tri-hydroxyalkylammonium salt.

The inhibition of fatty acid synthesis in biological systems by the useof garcinia acid or its derivatives is believed to arise from theinhibition by such compounds of citrate cleavage enzyme contained insuch systems. The cleavage of citrate is catalyzed by citrate cleavageenzyme according to the stoichiometry: citrate CoA ATP acetyl-CoAoxaloacetate ADP P,

In the conversion of carbohydrate and various amino acids to fat bynon-ruminant mammals, citrate is the major source of the acetyl group ofacetyl coenzyme A which is utilized for the synthesis of fatty acid.Citrate is formed in the mitochondria by the citrate synthase reaction.It is then metabolized via the citric acid cycle. Under conditions whenenergy intake exceeds energy demand, some citrate is diverted to theextramitochondrial space of the cell where it is used for fatty acidsynthesis, that is to say for energy storage.

Garcinia acid and its derivatives are useful in the treatment ofobesity. These compounds can be made up in the form of conventionalpharmaceutical preparations; for example, the aforesaid compounds can bemixed with conventional organic or inorganic inert pharmaceuticalcarriers suitable for parenteral or enteral administration such as, forexample, water, gelatin, lactose, starch, magnesium stearate, talc,vegetable oil, gums or the like. They can be administered inconventional pharmaceutical forms, e.g., solid forms, for example,tablets, dragees, capsules, suppositories or the like; or in liquidforms, for example, suspensions or emulsions. Moreover, thepharmaceutical composi-' tions containing compounds of this inventioncan be subjected to conventional pharmaceutical expedients such assterilization, and can contain conventional pharmaceutical excipientssuch as preservatives, stabilizing agents, emulsifying agents, salts forthe adjustment of osmotic pressure or buffers. The compositioncan alsocontain other therapeutically active materials.

A suitable pharmaceutical dosage unit can contain from about 15 to 600mg. of garcinia acid or its derivatives. Suitable parenteral dosageregimens in mammals comprise from 1 mg/kg to about 25 mg/kg per day.However, for any particular subject, the specific dosage regimen shouldbe adjusted according to individual need and the professional judgmentof the person administering or supervising the administration of theaforesaid compounds. It is to be understood that the dosages set forthherein are exemplary only and that they do not, to any extent, limit thescope or practice of this invention.

The present invention may be more clearly illustrated by the followingexamples. All temperatures are in degrees centrigrade.

EXAMPLE 1 Citrate cleavage enzyme was isolated from the liver of ratswhich had been starved for two days and which were then fed a diet highin glucose for three days. The purification was carried out using theprocedure of Inoue et al., J. Biochem. (Japan) 60, 543 (1966).

These steps involved ammonium sulfate precipitation between -30 percentsaturation and DEAIE-column chromatography.

The enzyme activity was measured as follows. The reaction mixturecontained 20 mM citrate, 20 mM magnesium chloride, 70 mM Tris-HCl buffer(pH 8.0), 200 mM hydroxylamine, mM dithiothreitol, 10 mM ATP, 0.6 mM CoAand citrate cleavage enzyme. The final volume was 1.0 ml. and thetemperature was 37 C. The reaction was started by adding the ATP. It wasstopped after 20 minutes and the hydroxamate color was developed asdescribed by Inoue et al. cited above. Hydroxamate formation was linearwith time for at least 30 minutes.

The above assay procedure was used for a series of tricarboxylic acidswhich were added to the reaction mixture in the amounts indicated inTable I which follows. The reaction mixture also contained 65 microgramsof protein. A millimolar extinction coefficient of 0.8 at 520millimicrons was used to calculate the amounts of hydroxamate formed.

TABLE I Citrate concentration (rnM) Substance added 10 Hydroxamateformed (mumoles/mg/rnin.) None 210 405 Homocitrate, 25 mM 6 52 310Homoisocitrate, 25 mM 12 190 40l Homoaconirate, 25 mM I2 198 385Garcinia acid, 1 mM 0 2 101 Table I indicates that garcinia acid is astrong inhibitor of citrate cleavage enzyme as evidenced by a lower rateof hydroxamate formation in the presence of this compound. The otheranalogs of citric acid which were tested produced much less inhibitioneven though they were used at twenty-five times the level of garciniaacid.

EXAMPLE 2 This example demonstrates the stereo-specific nature of thecitrate cleavage enzyme inhibition exhibited by garcinia acid. In thisexperiment the assay method described in Example 1 was utilized with theexception that garcinia acid and its stereoisomer (+)-allohydroxycitricacid were added in the amounts indicated below in Table 2.

TABLE 2 Citrate concentration (mM) Substance added 0.5 l0

Citrate cleaved (mumoleslmim) None 69 I65 Garcinia acid [0 ,uM 25 I44I00 M 6 96 (+)-Allohydroxy citric acid 0 prM 68 I62 I000 M 33 140 Asseen from the results summarized in Table 2, garcinia acid is a morepotent inhibitor of citrate cleavage enzyme than its structurallyrelated stereoisomer (+)-allohydroxy citric acid.

EXAMPLE 3 This example demonstrates the inhibition of lipogenesiseffected by treatment with garcinia acid in isolated rat liver slices.In these experiments Charles River female rats l50l75 gm. were fastedfor two days and refed ad libitum for three days on a diet containingpercent dextrose, 25 percent casein and 5 Phillips and Hart saltmixture.

The rats were killed by decapitation. Their livers were excised quickly,placed directly on ice for 30 seconds and 100-500 mg. slices were cutusing a modified Staty Riggs tissue slicer on ice. The serosal sliceswere discarded. Slices were transferred to 50 ml. beakers in icecontaining 10 times the tissue-volume of Hanks solutes dissolved in 5 mMTris. buffer pH 7.4-7.6. Fatty acid precursor in the form of alanine wasincluded along withthe transaminase acceptor a-keto glutaric acid inmolar ratio 2:3 at pH 7.4-7.6. Synthetic garcinia acid was added eitherin the lactone or free acid form in the final concentration indicatedbelow. Incubations were carried out at 37 C. under an atmosphere of 100percent oxygen in an Eberbach water bath shaker, usually for 60 minutesunless otherwise indicated. The incubation period began with theaddition of *C-alanine (specific activity= 107.2 mc/mM) to a finalconcentration of 10 ic/gm tissue. Two milliliters of 5N NaOI-I wereadded to 0" time control samples before C- alanine and appropriatecorrections made with the experimental samples.

Slices were removed with forceps, chilled directly on ice, added to 10ml. glass homogenizing tubes with 2 ml. of water and homogenized with 5strokes of a teflon pestle. Homogenates were transferred to tubescontaining 2 ml. 5N NaOH, and saponification was carried out for 3 hoursat 90 C. Samples were acidified with 2.5 ml. 5N H and extracted twicewith 5 ml. petroleum ether (boiling point 4060 C.). Supernatants wereadded directly to glass counting vials, evaporated to dryness and 10 ml.of toluene-PPO-POPOP scintillation fluid was added. Samples wereanalyzed for radioactivity in a Packard Tri-Carb scintillation counter.Data was expressed as rnpM of C-alanine incorporated/gram oftissue/60minutes.

In the first group of experiments liver slices were incubated for 60minutes with 10 mM *C-alanine and 15 mM a-ketoglutarate. Table 3illustrates the inhibition of lipogenesis by garcinia acid under theseconditions.

TABLE 3 Sample Additions mnmoleslgmlfifl" Inhibition 1 Control 167.5 2 511M Garcinia Acid 159.2 5.0 3 50 p.M Garc'mia Acid 118.9 29.0 4 500 nMGarcinia Acid 80.9 51.7

neutralized to pH 7.4

Table 4 demonstrates the inhibition by both garcinia acid and garciniaacid lactone in liver slices incubated for 60 minutes with 5 mM*C-alanine and 7.5 mM a-ketoglutaric acid. From these data thephysiological, apparent inhibition constant (K of garcinia acid was 350M and the K of garcinia acid lactone was 30 pLM.

neutralized to pH 7.4

Table 5 demonstrates the time kinetics of inhibition by garcinia acidunder the same experimental conditions observed in Table 4.

Table 5 Time (in mmnoles/ Sample Additions minutes) gm/t Inhibition 1Control 60 1 10.3 2 500 1.1M Garcinia Acid 60 83.7 24.1 3 Control 9239.6 4 500 pM Garcinia Acid 90 137.4 42.6 5 Control 120 737.4 6 500 11MGarcinia Acid* 120 248.1 66.3 neutralized to pH 7.4

EXAMPLE 4 This example demonstrates the in vivo activity of syntheticgarcinia acid and its lactone. Individual groups of Charles River femalerats 150-175 gm. were fasted for two days and meal fed from 9-12 a.m.using the diet .containing 70 percent dextrose, 25 percent casein and 5percent Philipps and Hart salt mixture.

0n the last day of refeeding, approximately 5 hours after feeding, theanimals were lightly anesthetized with Penthrane and injected (using a27 G needle) in the tail vein with the following composition: 12.6 mg.alanine as fatty acid precursor, 30.6 mg. a-ketoglutarate astransaminase acceptor and 5 ac C-alanine (specific activity 156 mc/mM)dissolved in a total volume of 0.25 ml. saline pH 7.4-7.6. Syntheticgarcinia acid either in the free acid or lactone form was dissolved in atotal volume of 0.25 ml. saline at pH 7.4-7.6.

The rats were killed by decapitation after 30 minutes. Livers wereexcised rapidly, weighed, placed in 30 ml. beakers with .15 ml. waterand crudely minced. They were transferred to glass homogenizing tubesand homogenized with 5 strokes of a teflon pestle. Three milliliteraliquots of liver homogenates in duplicate were added to tubescontaining 2.1 ml. 5N NaOH and saponified with 2.6 ml. 5N H 80 andextracted twice with 5 ml. of petroleum ether (boiling point 40-60 C.).Supernatants were added directly, to glass counting vials, evaporated todryness and 10 ml. of toluene-PPO- POPOP scintillation fluid added.Samples were analyzed for absolute activity in a Packard Tri-Carbscintillation counter. Resulting data was expressed as mumoles of*C-alanine incorporated/gram of tissue/30 minutes.

Fourteen rats were fasted for 2 days and meal fed as above for 3 days.On the last day of refeeding one group received 10 mg. of syntheticgarcinia acid in the lactone form dissolved in a total volume of 0.25ml. saline, pH 7.4-7.6 by tail vein injections 60 minutes prior toC-alanine. An additional 5 mg. of garcinia acid lactone was given withthe ,C-alanine. Control animals received only the C-alanine injection.Inhitibion of lipogenesis by garcinia acid lactone is illustrated inTable 6 below. In this table rats numbers 1-7 represent controls whilerats numbers 8-14 received garcinia acid lactone.

TABLE 6 (Garcinia Acid 83.5

Lactone) 66.8 S.E.M. 8.5 Inhibition compared with the controls numbers1-4.

TABLE 7 Rat No. (Controls) mnmoles/gm/30" 1048 S.E.M. 176.3 5 (GarciniaAcid Lactone) 386.1 6 249.1 7 290.9 8 257.8

296 S.E.M. i 31.4 Inhibition 72% EXAMPLE 5 A solution of (+)-garciniaacid lactone (10 g.) in tetrahydrofuran ml.) was treated with a solutionof diazomethane in ether until the yellow color persisted. The solutionwas allowed to stand at room temperature for one hour then the solventwas removed in vacuo and the residue crystallized from ether haxane togive 8.0 g. of (+)-garcinia acid lactone dimethyl ester, m.p. 70-72. Asecond crop (2.4 g.; m.p. 65-70) was obtained from the mother liquors.Crystallization from ether furnished the analytical sample, m.p. 72-73;[M 25 85.65 (c, 1.0, CHCl ir (CHCl 3550, 1810 and 1755 cm; nmr CDCl;,) 84.91 (singlet, 1H, CH), 4.14 (singlet, 1H, OH), 4.91 (singlet, 3H,-OCH;,), 3.78 (singlet, 3H, -OCH;,) and 2.99 (quartet, 2H, jCH

Anal. Calcd for C H O C, 44.04; H, 4.62. Found:

EXAMPLE 6 lowed to evaporate and the residue was dissolved in water (5ml.) and passed through a column of cation exchange resin (Amberlite IR10 ml.). The acidic eluent was evaporated to dryness to give a colorlesssolid. Crystallization from methanol-ethanol afforded 800 mg. of(+)-garcinia acid lactone mono ammonium salt, m.p. 231 (decomposition);[0 +929 (c, 1.0, H 0); 1r (KBr) 3,460-2,500 (broad), 1,800, 1,770 and1,625 (broad) cm ;nmr(DMSO)8 2.71 (quartet, 2H, CH and 4.60 (singlet,11-1, CH). Neutralization equivalent: 207 Anal. Calcd for C H NO C,34.79; H, 4.38; N,

Found: C, 34.65; H, 4.48; N, 6.72.

EXAMPLE 7 Acetyl chloride (3.0 ml.) was added to aboslute ethano] (50ml.), then after several minutes (+)-garcinia acid lactone (5.0 g.) wasadded and the solution heated under reflux for three hours. Molecularsieve 3A in a Sohxlet apparatus was used as a water scavenger. Thesolvent was removed in vacuo to give 75 g. of a pale yellow oil whichwas purified by reduced pressure distillation to give 5.2 g. of awater-white viscous liquid, b.p. 136-1400.1-0.15 mm; ir (CHCl 3,600,1,810 and 1,745 cm. Nmr analysis indicated that it was a 2:1 mixture of(+)-gar'cinia acid lactone diethyl ester and garcinia acid triethylester.

The triester was converted to the diester lactone upon repeateddistillation and pure diester lactone can be obtained in this manner.

EXAMPLE 8 Capsule Formulation Per Capsule Garcinia acid lactone 10 mgLactose, U.S.P. 165 mg Com Starch. U.S.P. 30 mg Talc. U.S.P. 5. mg TotalWeight 210 mg Procedure EXAMPLE 9 Capsule Formulation Per CapsuleGarcinia acid lactone 50 mg Lactose. U.S.P. 1 25 mg Corn Starch. U.S.P.30 mg Talc. USP. mg

Total Weight 210 mg Procedure 1. Garcinia acid lactone was mixed withlactose and corn starch in a suitable mixer.

2. The mixture was further blended by passing through a FitzpatrickComminuting Machine with a 1A screen with knives forward.

3. The blended powder was returned to the mixer, the talc added andblended thoroughly.

4. The mixture was filled into 4 hard shell gelatin capsules on a ParkeDavis capsulating machine.

EXAMPLE 10 Tablet Formulation Per Tablet Garcinia acid lactone 25.00 mgDicalcium Phosphate Dihydrate, Unmilled 175.00 mg Corn Starch 24.00 mgMagnesium Stearate 1.00 mg Total Weight 225.00 mg Procedure 1. Garciniaacid lactone and corn starch were mixed together and passed through an00 screen in Model J" Fitzmill with hammers foward.

2. This premix was then mixed with dicalcium phosphate and onehalf ofthe magnesium stearate, passed through a 1A screen in Model .1 Fitzmillwith knives EXAMPLE ll Tablet Formulation Per Tablet Garcinia acidlactone 100 mg Lactose, U.S.P. 202 mg Corn Starch, U.S.P. mg Amijel B01120 mg Calcium Stearate 8 mg Total Weight 410 mg A prehydrolyzed foodgrade corn starch. Any similar prehydrolyzed corn starch may be useProcedure 1. Garcinia acid lactone, lactose, corn starch, and AmijelB011 were blended in a suitable mixer.

2. The mixture was granulated to a heavy paste with water and the moistmass was passed through a 12 screen. It was then dried overnight at 1 10F.

3. The dried granules were passed through a 16 screen and transferred toa suitable mixer. The calcium stearate was added and mixed untiluniform.

4. The mixture was compressed at a tablet weight of 410 mg. using tabletpunches having a diameter of approximately three-eight inch. (Tabletsmay be either flat or biconvex and may be scored if desired.)

I claim:

1. A method for treating obesity which comprises administering to amammal in need of such treatment an effective amount of a compoundselected from the group consisting of garcinia acid, garcinia acidlactone, mono-, diand trilower alkyl, phenyl and benzyl esters ofgarcinia acid, monoand di-lower alkyl, phenyl and benzyl esters ofgarcinia acid lactone, wherein lower alkyl is from one to seven carbonatoms, and non-toxic pharmaceutically acceptable basic salts thereof.

2. The method of claim 1 wherein garcinia acid is administered.

3. The method of claim 1 wherein garcinia acid lactone is administered.

4. The method of claim I wherein anester of garcinia acid or garcinialactone is administered.

- 5. The method of claim 4 wherein said ester is a lower alkyl ester.

6. The method of claim 1 wherein the compound is administered in therange of from about 1 to about 25 mg/kg per day.

7. A pharmaceutical composition for the treatment of obesity comprisinga pharmaceutical carrier and an effective amount of a compound selectedfrom the group consisting of garcinia acid, garcinia acid lactone,mono-, diand tri-lower alkyl, phenyl and benzyl esters of garcinia acid,monoand di-lower alkyl, phenyl and benzyl esters of garcinia acidlactone, wherein lower alkyl is from one to seven carbon atoms, andnon-toxic pharmaceutically acceptable basic salts thereof.

8. The composition of claim 7 wherein said compound is garcinia acid.

9. The composition of claim 7 wherein said compound is garcinia acidlactone.

10. The composition of claim 8 wherein said compound is an ester ofgarcinia acid or garcinia acid lactone.

11. The composition of claim 10 wherein said ester is a lower alkylester.

12. The composition of claim 7 wherein said compound is present in therange of from about 15 to 600 mg.

2. The method of claim 1 wherein garcinia acid is administered.
 3. Themethod of claim 1 wherein garcinia acid lactone is administered.
 4. Themethod of claim 1 wherein an ester of garcinia acid or garcinia lactoneis administered.
 5. The method of claim 4 wherein said ester is a loweralkyl ester.
 6. The method of claim 1 wherein the compound isadministered in the range of from about 1 to about 25 mg/kg per day. 7.A pharmaceutical composition for the treatment of obesity comprising apharmaceutical carrier and an effective amount of a compound selectedfrom the group consisting of garcinia acid, garcinia acid lactone,mono-, di- and tri-lower alkyl, phenyl and benzyl esters of garciniaacid, mono- and di-lower alkyl, phenyl and benzyl esters of garciniaacid lactone, wherein lower alkyl is from one to seven carbon atoms, andnon-toxic pharmaceutically acceptable basic salts thereof.
 8. Thecomposition of claim 7 wherein said compound is garcinia acid.
 9. Thecomposition of claim 7 wherein said compound is garcinia acid lactone.10. The composition of claim 8 wherein said compound is an ester ofgarcinia acid or garcinia acid lactone.
 11. The composition of claim 10wherein said ester is a lower alkyl ester.
 12. The composition of claim7 wherein said compound is present in the range of from about 15 to 600mg.